Project Summary/Abstract The goal of this project is to develop accessible and effective methods to monitor the levels and interaction partners of GlcNAc-containing glycoconjugates. N-acetylglucosamine (GlcNAc) is a monosaccharide found in many classes of mammalian glycoconjugates. Addition of GlcNAc to serine and threonine residues forms the intracellular O-GlcNAc modification, and GlcNAc is also incorporated into many extracellular glycoconjugates including N-linked glycans and GalNAc-type glycans. Incorporation of GlcNAc into glycoconjugates is catalyzed by the activity of N-acetylglucosaminyltransferases (GlcNAc-transferases) that transfer GlcNAc from the donor UDP-GlcNAc to (glyco)protein and (glyco)lipid acceptors. UDP-GlcNAc is produced through the nutrient-sensing hexosamine biosynthetic pathway, which integrates information about carbohydrate, protein, lipid, and nucleotide availability. UDP-GlcNAc levels regulate production of key glycan structures, namely O- GlcNAcylation and N-linked glycan branching, which in turn control essential signal transduction pathways. Thus, GlcNAc-containing glycans represent a crucial link between metabolic state and cellular signaling. However, cell-based methods to characterize the levels and interaction partners of these molecules remain inadequate. Aim 1 will deliver non-invasive, non-perturbing fluorescent and luminescent reporters of intracellular O-GlcNAc levels. This Aim builds on the discovery that splicing of the O-GlcNAc transferase (OGT) transcript responds rapidly to changes in O-GlcNAc levels. Aims 2-4 improve upon previously reported photocrosslinking sugar technology, in which the diazirine photoactivatable crosslinking group is installed on GlcNAc residues in living cells. Aim 2 will make this technology easier to use by simplifying the reagents, improving crosslinking yield, and facilitating purification of crosslinked complexes. Aim 3 will make this technology broader in scope by introducing photocrosslinking GlcNAc into additional classes of glycoconjugates, including N-linked glycans. Aim 4 will make the technology more powerful by developing a mass spectrometry strategy to identify not only the identity of the sites of the binding partners, but also the sites of crosslinking. The mass spectrometry-based approach to crosslinking analysis will capture molecular details of O-GlcNAc-dependent interactions that occur in living cells. The reagents and methods developed in this proposal will be shared with other research groups to enable study of a wide variety of O-GlcNAcylation and N-glycosylated proteins with diverse biological functions. The proposed work prioritizes approaches that are simple to implement and make use of ?off-the-shelf? reagents and procedures. Making these methods available to the broad biomedical community is significant because dysregulation of GlcNAc-containing glycoconjugates is associated with multiple disease states including diabetes, neurodegenerative disease, and cancer.